Sickle cell disease is one of the most commonly inherited blood disorders among Americans of Hispanic and African descent. Sickle red cells are more prone to hemolysis than normal red cells, resulting in increased levels of plasma hemoglobin, a known pro-oxidant. The vascular complications are likely due to this increase in oxidative stress. The long-term objective of this project is to understand the relationship between hemolysis, lipid oxidation and vascular dysfunction in sickle cell disease. This objective will be tested using the following Specific Aims: 1) Measure the level and oxidation state of plasma hemoglobin using novel techniques, a) Plasma hemoglobin levels and oxidation state will be determined using electron paramagnetic resonance spectroscopy. b) Hydroethidine will serve as a probe to detect plasma hemoglobin through a one-electron oxidation of HE in normal and sickle plasma and products resolving using HPLC techniques. 2) Measure oxidative products and the potential to support lipid oxidation in normal and sickle plasma, a) F2-isoprostanes, a vaso-constrictive lipid oxidation product, will be compared in both sickle cell and normal control plasma using liquid chromatography and mass spectrometry. b) The oxidation potential of sickle cell and normal plasma will be measured using an oxygen electrode-based method, c) The degree of correlation between these measurements and plasma hemoglobin levels will be examined. 3) Examine the relationship between hemolysis and vascular dysfunction using histological and vascular response assays in transgenic mouse models, a) Mouse plasma hemoglobin levels will be measured using EPR spectroscopy and an HE oxidation assay, b) Plasma F2-isoprostane levels and other oxidation potential will be measured using methods developed above; c) Measure vessel responsiveness using a facialis artery preparation; d) Analyze nitrotyrosine levels by western blot and immunohistochemistry; e) Examine histological changes using hematoxylin and eosin (H&E). The RELEVANCE to health in the proposed research project is gaining a more complete understanding of the role of hemoglobin and oxidative damage in the pathophysiology of sickle cell disease. Current treatments for an acute sickle cell crisis are mainly palliative, as patients seek medical care when complications present themselves. When completed, this research should lead to the discovery and exploitation of a new therapeutic target to aid in the treatment of sickle cell disease.